Laboratory for Molecular Pharmacology, Department of Neuroscience and Pharmacology, University of Copenhagen, Blegdamsvej 3b, DK-2200, Copenhagen, Denmark.
J Biol Chem. 2011 Jun 10;286(23):20845-60. doi: 10.1074/jbc.M110.173237. Epub 2011 Mar 14.
Based on the conformationally constrained D-Trp-Phe-D-Trp (wFw) core of the prototype inverse agonist [D-Arg(1),D-Phe(5),D-Trp(7,9),Leu(11)]substance P, a series of novel, small, peptide-mimetic agonists for the ghrelin receptor were generated. By using various simple, ring-constrained spacers connecting the D-Trp-Phe-D-Trp motif with the important C-terminal carboxyamide group, 40 nm agonism potency was obtained and also in one case (wFw-Isn-NH(2), where Isn is isonipecotic acid) ~80% efficacy. However, in contrast to all previously reported ghrelin receptor agonists, the piperidine-constrained wFw-Isn-NH(2) was found to be a functionally biased agonist. Thus, wFw-Isn-NH(2) mediated potent and efficacious signaling through the Gα(q) and ERK1/2 signaling pathways, but in contrast to all previous ghrelin receptor agonists it did not signal through the serum response element, conceivably the Gα(12/13) pathway. The recognition pattern of wFw-Isn-NH(2) with the ghrelin receptor also differed significantly from that of all previously characterized unbiased agonists. Most importantly, wFw-Isn-NH(2) was not dependent on GluIII:09 (Glu3.33), which otherwise is an obligatory TM III anchor point residue for ghrelin agonists. Molecular modeling and docking experiments indicated that wFw-Isn-NH(2) binds in the classical agonist binding site between the extracellular segments of TMs III, VI, and VII, interacting closely with the aromatic cluster between TMs VI and VII, but that it does so in an opposite orientation as compared with, for example, the wFw peptide agonists. It is concluded that the novel peptide-mimetic ligand wFw-Isn-NH(2) is a biased ghrelin receptor agonist and that the selective signaling pattern presumably is due to its unique receptor recognition pattern lacking interaction with key residues especially in TM III.
基于原型反向激动剂 [D-Arg(1),D-Phe(5),D-Trp(7,9),Leu(11)] 神经肽的构象受限 D-Trp-Phe-D-Trp (wFw) 核心，生成了一系列新型的、小的、肽模拟激动剂用于胃饥饿素受体。通过使用各种简单的、环约束的间隔物将 D-Trp-Phe-D-Trp 基序与重要的 C 末端羧酰胺基连接，获得了 40nm 的激动活性，并且在一种情况下（wFw-Isn-NH(2)，其中 Isn 是异哌啶酸）获得了~80%的效力。然而，与所有先前报道的胃饥饿素受体激动剂不同，被约束的哌啶基 wFw-Isn-NH(2) 被发现是一种功能偏向激动剂。因此，wFw-Isn-NH(2) 通过 Gα(q) 和 ERK1/2 信号通路介导有效的信号转导，但与所有先前的胃饥饿素受体激动剂不同，它不通过血清反应元件（血清反应元件）信号转导，推测通过 Gα(12/13) 途径。wFw-Isn-NH(2) 与胃饥饿素受体的识别模式也与所有先前表征的无偏激动剂显著不同。最重要的是，wFw-Isn-NH(2) 不依赖于 GluIII:09（Glu3.33），否则它是胃饥饿素激动剂的必需 TM III 锚定位点残基。分子建模和对接实验表明，wFw-Isn-NH(2) 结合在 TM III、VI 和 VII 的细胞外片段之间的经典激动剂结合位点，与 TM VI 和 VII 之间的芳香族簇紧密相互作用，但与例如 wFw 肽激动剂相比，它以相反的方向结合。结论是，新型肽模拟配体 wFw-Isn-NH(2) 是一种偏向的胃饥饿素受体激动剂，选择性信号转导模式推测是由于其独特的受体识别模式，缺乏与关键残基的相互作用，特别是在 TM III 中。
